Anthrax toxin (AT) is crucial for the lethality of Bacillus anthracis, the causative agent of anthrax. The toxin comprises three secreted proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Alone, none of the proteins is toxic to cells or animals, but PA+EF and PA+LF are toxic. EF is an adenylyl cyclase that harms cells by elevating cAMP. LF is a Zn-dependent metalloprotease that cleaves mitogen activated protein kinase kinases (MAPKKs). PA itself is nontoxic, but is crucial because it delivers EF and LF to cytosol by receptor-mediated endocytosis. Two PA receptors are known, tumor endothelial marker 8 and capillary morphogenesis gene protein 2. Toxin entry into cells comprises multiple steps: PA binds receptors, furin cleaves off a portion of PA, bound PA forms a heptamer, EF and LF bind the heptamer, the whole complex is internalized, the toxin is routed to endosomes, and EF and LF enter cytosol via the PA heptameric channel. The toxin's action in cytosol results in yet another set of events that culminate in harm to the cell, even lethality. Thus, anthrax toxin action on cells spans a complex cascade of events, which clearly requires normal functioning of many cellular proteins. This proposal aims to identify and characterize novel genes whose products anthrax toxin requires to intoxicate cells. The central hypothesis is that normal functioning of certain cellular genes is crucial for intoxication and that disruption of such genes would disrupt the toxin's action on cells. The proposal has four specific aims: 1) development of toxin resistant clones. This will be done by random, global disruption of gene functions with various techniques, followed by selection of toxin resistant clones. 2) identification of disrupted genes. 3) assessment of candidate genes'relevance to toxin resistance. 4) detailed analysis of those genes that prove relevant to intoxication. Anthrax bacteria can be eliminated effectively with antibiotics at various stages of infection. However, such treatment must begin early. At later stages, usually 2 or 3 days following appearance of bacteria in blood, bacteria can still be eliminated, but they leave enough toxin behind to kill the host. Thus, treatment strategies at such stages must include disruption of the toxin's action on cells. To that end, identification of new genes and their role in aiding anthrax toxin action will facilitate devising strategies to block the toxin's action.